Prostaglandin E2 Stimulates Fibronectin Expression through EP1 Receptor, Phospholipase C, Protein Kinase Cα, and c-Src Pathway in Primary Cultured Rat Osteoblasts*

Fibronectin (Fn) is involved in the early stages of bone formation, and prostaglandin E (PGE) is an important factor regulating osteogenesis. Here we found that PGE2 enhanced extracellular Fn assembly in rat primary osteoblasts, as shown by immunofluorescence staining and enzyme-linked immunosorbent assay. PGE2 also increased the protein levels of Fn by using Western blotting analysis. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptor, antisense oligonucleotides revealed that the EP1 receptor but not other PGE receptors is involved in PGE2-mediated up-regulation of Fn. At the mechanistic level, Ca2+ chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)), phosphatidylinositol-phospholipase C inhibitor (U73122), or Src inhibitor (PP2) attenuated the PGE2-induced Fn expression. Protein kinase C (PKC) inhibitor (GF109203X) also inhibited the potentiating action of PGE2. Furthermore, treatment with antisense oligonucleotides of various PKC isoforms, including α, β, ϵ, and δ, demonstrated that α isozyme plays an important role in the enhancement action of PGE2 on Fn assembly. Flow cytometry and reverse transcription-PCR showed that PGE2 and 17-phenyl trinor PGE2 (EP1/EP3 agonist) increased the surface expression and mRNA level of α5 or β1 integrins. Fn promoter activity was enhanced by PGE2 and 17-phenyl trinor PGE2 in cells transfected with pGL2F1900-Luc. Cotransfection with dominant negative mutants of PKCα or c-Src inhibited the potentiating action of PGE2 on Fn promoter activity. Local administration of PGE2 or 17-phenyl trinor PGE2 into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the Fn and α5β1 integrin immunostaining and bone volume of secondary spongiosa in tibia. Taken together, our results provided evidence that PGE2 increased Fn and promoted bone formation in rat osteoblasts via the EP1/phospholipase C/PKCα/c-Src signaling pathway.

The extracellular matrix (ECM) 1 provides positional and environmental information that is essential for tissue function.
The ECMs produced by osteoblasts are complex and consist of several different classes of molecules that may regulate the modeling and remodeling of bone. The ECMs also serve as a reservoir for growth factors, including members of the prostaglandins (PGEs) and fibroblast growth factor superfamily (1,2). Acting either alone or together, these components of the ECM produced by osteoblasts may subsequently regulate the cell adhesion, migration, proliferation, differentiation, survival, as well as the rate of bone formation.
Fibronectin (Fn) is an extracellular matrix component that is also present as a soluble protein in plasma and other body fluids (3). The matrix form of Fn is believed to support cell adhesion and migration during embryogenesis, tumor growth, wound healing, angiogenesis, and inflammation (4). Assembly of soluble Fn into matrix is a multistep process under cellular control (5). Among the membrane components implicated in Fn matrix assembly, integrins have been demonstrated to have a central role (6). Integrins, composed of ␣ and ␤ subunits, are a family of transmembrane receptors mediating adhesion to both ECM and cell surface molecules (7,8). The specific adhesion depends on the interaction between the cell-binding domain of Fn and cell surface integrin receptors. However, the mechanisms regarding how integrins modulate Fn assembly are not well understood. Transfection of ␣5 integrin and expression of ␣5␤1 integrin by Chinese hamster ovary cells results in a large increase in Fn assembly, whereas ␣5-deficient Chinese hamster ovary B2 cells failed to assemble plasma Fn into the ECM (9,10). Osteoblast differentiation is an essential part of bone formation, because active osteoblasts should be recruited at the site of osteoclastic bone resorption to compensate for the continuous loss of bone matrix and to maintain the structural integrity of the skeletal system. The biology of this process is also of considerable interest when applying therapies to promote bone repair after injury or during disease processes. Furthermore, integrins are involved in the signal transduction of translating the strain in the organic matrix to the biochemical signals in the bone cells (11). However, the role of cytokine in the cell-matrix interactions in osteoblasts has not been extensively studied.
PGEs are considered important local factors that modulate bone metabolism through their effects on osteoblastic cells and osteoclasts (12). PGE 2 is a major eicosanoid produced by osteoblasts. To explain the diverse effects of PGE 2 , the presence of multiple receptors for PGE 2 in osteoblasts was postulated. Recent cloning of four subtypes of PGE receptor has made it possible to analyze the PGE receptor subtypes (EP 1 -EP 4 ) on osteoblasts (13,14). EP 1 is coupled to Ca 2ϩ mobilization; EP 2 and EP 4 activate adenylate cyclase, and EP 3 inhibits adenylate cyclase (15)(16)(17). An EP 1 agonist stimulated cell growth, whereas an EP 4 agonist reduced cell growth and increased alkaline phosphatase activity in MC3T3-E1 osteoblast-like cells (18). These studies indicate that osteoblasts express multiple subtypes of the PGE receptor and that each subtype might be linked to different actions of PGE 2 .
The distribution of Fn in areas of skeletogenesis suggests that it may be involved in early stages of bone formation (19). However, the effect of PGE 2 on Fn fibrillogenesis in osteoblasts is mostly unknown. Here we found that PGE 2 enhanced Fn fibrillogenesis of osteoblasts by increasing the synthesis and assembly of Fn. Furthermore, the increase of clustering of ␣5 and ␤1 integrins is involved in the action mechanism of PGE 2 . EP 1 receptor, PI-PLC, PKC␣, and c-Src-dependent pathways may be involved in the increase of osteoblast Fn expression and bone formation by PGE 2 .

EXPERIMENTAL PROCEDURES
Materials-Mouse monoclonal antibody for PKC␣ was purchased from BD Transduction Laboratories. Mouse monoclonal antibody for ␣-tubulin was purchased from Oncogene Science (Cambridge, MA). Protein-A/G beads, anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for fibronectin, phosphotyrosine residues (PY20), and c-Src were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies specific for ␣5, ␤1, and ␣5␤1 integrin and type I collagen were purchased from Chemicon (Temecula, CA). PGE 2 , 17-phenyl trinor PGE 2 , butaprost, sulprostone, 11-deoxy-PGE 1 , and SC19220 were purchased from Cayman Chemical (Ann Arbor, MI). U73122, U73343, D609, and GF109203X were purchased from Calbiochem. Avidin-biotin-peroxidase detection system was purchased from Vector Laboratories. The fibronectin promoter construct (pGL2F1900-Luc) was a gift from Dr. I. S. Kim (Kyungpook National University, Korea). The PKC␣ dominant negative mutant was a gift from Dr. V. Martin (Louis Pasteur de Strasbourg University, France). The c-Src dominant negative mutant was a gift from Dr. S. Parsons (University of Virginia Health System, Charlottesville, VA). pSV-␤-galactosidase vector and luciferase assay kit were purchased from Promega (Madison, MA). All other chemicals were obtained from Sigma.
Primary Osteoblast Cultures-Primary osteoblastic cells were prepared by the method described previously (20). The calvaria of fetal rats were dissected from fetal rats, divided into small pieces, and then treated with 0.1% type I collagenase solution for 10 min at 37°C. The next two 20-min sequential collagenase digestions were then pooled and filtered through 70-m nylon filters (Falcon). The cells were grown on the plastic cell culture dishes in 95% air, 5% CO 2 with ␣-minimum Eagle's medium (Invitrogen) that was supplemented with 20 mM HEPES and 10% heat-inactivated fetal calf serum, 2 mM-glutamine, penicillin (100 units/ml), and streptomycin (100 g/ml) (pH adjusted to 7.6). The characteristics of osteoblasts were confirmed by morphology and the expression of alkaline phosphatase.
Immunocytochemistry-Osteoblasts were grown on glass coverslips. Cultures were rinsed once with phosphate-buffered saline (PBS) and fixed for 15 min at room temperature in phosphate buffer containing 4% paraformaldehyde. Cells were then rinsed three times with PBS. After blocking with 4% BSA for 15 min, cells were incubated with rabbit anti-rat Fn (1:1000) for 1 h at room temperature. Cells were then washed again and labeled with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:150, Leinco Technologies, St. Louis, MO) for 1 h. Finally, cells were washed, mounted, and examined with a Zeiss confocal microscope (LSM 410) as soon as possible. The mean fluorescence under 10 -15 cells (3-5 fields per culture) was measured by using a Zeiss confocal microscope. The focus of the z axis was on the substratum of the monolayer cells. The value for contrast and offset adjustment of confocal microscope was fixed so that the variation of the relative fluorescence of control experiments was rather small.
Quantification of Extracellular Immobilized Fn by ELISA-The level of extracellular immobilized Fn was also determined by an enzymelinked immunosorbent assay (ELISA). After treatment with PGE 2 at 37°C, the cells were washed twice with PBS and fixed at room temperature with 1% paraformaldehyde for 30 min. After washing with PBS, the cultures were then blocked with 1% BSA in PBS for 15 min before being incubated sequentially with rabbit anti-rat Fn antibody (1:150) for 1 h and horseradish peroxidase-labeled anti-rabbit antibody (1: 1000) for 30 min. After each incubation, the cells were washed two times with PBS. o-Phenylenediamine dihydrochloride substrate (0.4 mg/ml in phosphate/citrate buffer, pH 5.0; 24.3 mM citric acid; 51.4 mM Na 2 HPO 4 ⅐12 H 2 O; 12% H 2 O 2 (v/v)) was then applied to the cells for 30 min, and 3 M sulfuric acid was added to stop the reaction. The absorbance was measured at 450 nm by an ELISA reader (Bio-Tek, Burlington, VA). Each assay was performed in triplicate.
Immunoprecipitation and Western Blot Analysis-The cellular lysates were prepared as described previously (20). Equal amounts of protein were incubated with specific antibody immobilized onto protein-A/G-Sepharose for 12 h at 4°C with gentle rotation. Beads were washed extensively with lysis buffer, boiled, and microcentrifuged. Proteins were resolved on SDS-PAGE and transferred to Immobilon polyvinylidene difluoride membranes. The blots were blocked with 4% BSA for 1 h at room temperature and then probed with rabbit anti-rat antibodies against Fn (1:1500) or c-Src (1:1000) for 1 h at room temperature. After three washes, the blots were subsequently incubated with a donkey anti-rabbit peroxidase-conjugated secondary antibody (1:1000) for 1 h at room temperature. The blots were visualized by enhanced chemiluminescence using Kodak X-Omat LS film (Eastman Kodak Co.). For normalization purposes, the same blot was also probed with mouse anti-rat ␣-tubulin antibody (1:1000). Quantitative data were obtained by using a computing densitometer and ImageQuant software (Amersham Biosciences).
Determination of Cytosolic Ca 2ϩ with Fluo-3-AM-Fluo-3-acetoxymethyl ester (fluo-3-AM) was used to measure cytosolic free Ca 2ϩ . Cells were incubated for 60 min in the dark at room temperature with fluo-3-AM (4 M), and the cells were then washed, and cytosolic Ca 2ϩ was measured by FACSCalibur (CellQuest software, BD Biosciences). Excitation and emission wavelengths were 488 and 530 nm, respectively.
Quantification of Integrin Expression-Osteoblasts were plated in 6-well (35-mm) dishes. The cells were then washed with PBS and detached with trypsin at 37°C. Cells were fixed for 10 min in PBS containing 1% paraformaldehyde. After rinsing in PBS, the cells were incubated with rabbit anti-rat ␣5 or ␤1 integrin antibody (1:100) for 1 h at 4°C. Cells were then washed again, incubated with fluorescein isothiocyanate-conjugated secondary IgG for 45 min, and analyzed by flow cytometry using FACSCalibur.
Transfection and Reporter Gene Assay-Osteoblasts were cotransfected with 1 g of Fn promoter plasmid and 1 g of ␤-galactosidase expression vector. Osteoblasts were grown to 60% confluence in 12-well plates and were transfected the following day by LF2000, premixed DNA with OPTI-MEM, and LF2000 with OPTI-MEM, respectively, for 5 min. The mixture was then incubated for 25 min at room temperature and added to each well. After a 24-h incubation, transfection was complete, and the cells were incubated with the indicated agents. After 24 h of incubation, the media were removed, and cells were washed once with cold PBS. To prepare lysates, 100 l of reporter lysis buffer (Promega, Madison, WI) was added to each well, and cells were scraped from dishes. The supernatant was collected after centrifugation at 13,000 rpm for 30 s. Aliquots of cell lysates (10 l) containing equal amounts of protein (10 -20 g) were placed into wells of an opaque black 96-well microplate. An equal volume of luciferase substrate was added to all samples, and luminescence was measured in a microplate luminometer. The luciferase activity value was normalized to transfection efficiency monitored by the cotransfected ␤-galactosidase expression vector. In experiments using dominant negative mutants, cells were cotransfected with reporter (0.5 g) and ␤-galactosidase (0.25 g) and either the PKC␣ or c-Src mutant or the empty vector (1.0 g).
Measurement of Bone Mineral Density (BMD) and Bone Volume-The local injection of young rats was prepared by the method described previously (23). Male Sprague-Dawley rats weighing 73-88 g were used. Implantation of a cannula (22-gauge) was done from the posterolateral side into the proximal tibial metaphysis in both limbs of rats anesthetized Total RNA was extracted from primary rat osteoblastic cells, and subjected to RT-PCR for EP 1 , EP 2 , EP 3 , and EP 4 mRNAs using the respective primers. Note that primary rat osteoblasts express EP 1 -EP 4 receptor mRNA, and EP 1 mRNA increased in response to PGE 2 (3 M) application for 6 h (A). Osteoblasts were transfected with EP receptor AS-ODN or MM-ODN for 24 h followed by incubation with PGE 2 for 6 and 24 h to analyze the mRNA and protein expression, respectively. Total protein and RNA were isolated, and the expressions of Fn and EP receptors were analyzed by Western blotting (WB) and RT-PCR (RT) (B). Results are representative of at least three independent experiments. GAPDH, glyceraldehyde-3phosphate dehydrogenase.
with trichloroacetaldehyde. The cannula had its outer end in the subcutaneous tissue. PGE 2 or 17-phenyl trinor PGE 2 (30 M, 10 l) was percutaneously injected into the proximal tibia through the cannula (once/ day) for 7 consecutive days. The same volume of vehicle was injected into the contralateral side for comparison. On day 14, the rats were sacrificed, and the tibiae were also removed and cleaned of soft tissue. BMD and BMC of the tibia were measured with a dual-energy x-ray absorptiometer (DEXA, XR-26; Norland, Fort Atkinson, WI). The mode adapted to the measurements of small subjects was adopted. A coefficient of variation of 0.7% was calculated from daily measurements of BMD on a lumbar phantom for more than 1 year. The whole tibiae were scanned, and BMD and BMC were measured by absorptiometer. At the end of the program, the tibia was fixed, decalcified, and embedded in paraffin. Serial sections (5 m) were cut longitudinally, and endogenous peroxidase activity was inactivated by treatment with 3% H 2 O 2 in methanol for 20 min. The sections were then treated with normal goat serum to block nonspecific binding, followed by incubation with rabbit anti-rat Fn, ␣5␤1 integrin, and type I collagen antibody (1:300) overnight at 4°C. The sections were detected by avidin-biotin-peroxidase detection system and diaminobenzidine. For measurement of bone volume, the sections were stained with Mayer's hematoxylin and eosin solution. Images of the growth plate and proximal tibia were photographed by using an Olympus microscope IX70. Measurement of bone volume was performed on the secondary spongiosa, which is located 1.0 -3.0 mm distal to epiphyseal growth plate and is characterized by a network of larger trabeculae. Bone volume was calculated using image analysis software (Image-Pro Plus 3.0) and expressed as percent of bone area. All measurements were done in a single-blind fashion. All protocols complied with institutional guidelines and were approved by Animal Care Committee of Medical College, National Taiwan University. Statistics-The values given are means Ϯ S.E. The significance of difference between the experimental groups and controls was assessed by Student's t test. The difference is significant if the p value was Ͻ0.05.

PGE 2 Enhanced Fn Fibrillogenesis in Cultured Osteoblasts-
The fibrillogenesis from the endogenously released Fn by the primary cultured rat osteoblasts was studied using immunocytochemistry. Day 3-5 osteoblasts were changed to serumfree medium and incubated with PGE 2 (3 M) for 24 h. Immunostaining of Fn was examined in 4% paraformaldehyde-fixed and nonpermeabilized cells. The mean immunofluorescence intensity underneath a cell group of 10 -15 cells was measured using a confocal microscope. As shown in Fig.  1A, osteoblasts are able to form Fn network underneath the cell using endogenously released Fn. Fn fibril formation increased in response to the treatment of PGE 2 for 24 h (Fig.  1B). The quantitative data showed a dose-dependent increase of fluorescence intensity (Fig. 1C). We also used ELISA to detect extracellular immobilized Fn. PGE 2 also increased Fn expression in a concentration-dependent manner (Fig. 1D). Western blotting was used to examine the effect of PGE 2 on the protein levels of Fn. Day 3-5 osteoblasts were changed to serum-free culture medium and treated with PGE 2 for 24 h. The cultures were then washed with cold PBS, and protein samples were collected by the addition of lysis buffer without trypsin digestion. The result from Western blotting may contain both soluble cytosolic Fn and extracellular immobilized Fn. As shown in Fig. 1, E and F (PGE 2 at 3 M), PGE 2 increased protein levels of Fn in a concentration-and timedependent manner.
Involvement of EP 1 Receptors in PGE 2 -mediated Increase of Fn Formation-PGEs exert their effects through interaction with specific EP 1-4 receptors (14). To investigate the role of EP 1-4 subtype receptors in PGE 2 -mediated increase of Fn formation, we assessed the distribution of these EP subtype re-ceptors in rat primary osteoblasts by RT-PCR analysis. The mRNAs of EP 1 , EP 2 , EP 3 , and EP 4 subtype receptors could be detected in primary rat osteoblasts ( Fig. 2A). After PGE 2 treatment for 6 h, the mRNA level of EP 1 subtype receptor was evidently increased, whereas other subtype EP receptor mRNAs remained unchanged ( Fig. 2A). We next examined which EP subtype receptors were involved in the PGE 2 -mediated increase of Fn formation, and specific inhibition of EP 1 receptor expression was accomplished with AS-ODN. It was found that EP 1 receptor-specific AS-ODN but not other EP receptor AS-ODN or MM-ODN significantly blocked the PGE 2mediated increase of Fn formation in primary rat osteoblasts (Fig. 2B). To determine the role of EP 1 receptor-dependent signaling in the regulation of Fn expression in osteoblasts, the cells were treated with EP 1-4 -specific agonists, and then the expression level of Fn was examined. Of the agonists tested, only the EP 1 /EP 3 -selective receptor agonist, 17-phenyl trinor PGE 2 (3 M), significantly increased the protein level of Fn (Fig. 3A). In contrast, butaprost (EP 2 agonist; 10 M), sulprostone (EP 3 agonist; 10 M), and 11-deoxy-PGE 1 (EP 2 /EP 4 -selective agonist; 10 M) failed to up-regulate Fn expression. In addition, treatment of EP 1 receptor antagonist SC19220 (10 M) effectively antagonized the potentiating effect of PGE 2 on Fn expression (Fig. 3A). It has been reported that sulprostone also acts on the rat EP 1 receptor (26). We then examined the concentration-dependent effect of sulprostone on the expression of Fn. Treatment of osteoblast with sulprostone did not increase the protein level of Fn unless at a higher concentration of 20 M. Pretreatment of osteoblasts with EP 1 AS-ODN but not EP 3 AS-ODN antagonized the potentiating action of 20 M sulprostone (Fig. 3B). The results shown above using pharmacological treatment or genetic inhibition clearly demonstrated a critical role for the EP 1 receptor in the PGE 2 -mediated increase of Fn formation. It has been reported that activation of EP 1 augments intracellular calcium mobilization, which is re- lated to downstream signals (15). We then investigated the effect of chelating intracellular Ca 2ϩ on the potentiating action of PGE 2 on Fn expression. Pretreatment with BAPTA-AM (0.1-10 M) for 30 min significantly abrogated PGE 2 -induced Fn formation (Fig. 3C). The quantitative data are shown in Fig. 3C, lower panels. Flow cytometry was used to investigate the effect of PGE 2 on the change of intracellular Ca 2ϩ concentration. As shown in Fig. 3D, incubation with PGE2 (3 M), 17-phenyl trinor PGE 2 (3 M), and sulprostone (20 M) enhanced the fluorescence intensity of fluo-3. However, sulprostone at 10 M only slightly increased the intracellular Ca 2ϩ concentration. ELISA detection also showed that pretreatment of osteoblasts with the EP 1 AS-ODN, SC19220, and BAPTA-AM but not AS-ODN of EP 2 -EP 4 or any MM-ODN antagonized the potentiating effect of PGE 2 (Fig. 3E).
The Signaling Pathways of PI-PLC, PKC, and c-Src Are Involved in the Potentiating Action of PGE 2 -To study the intracellular signaling pathway involved in PGE 2 -induced Fn expression, osteoblasts were pretreated for 30 min with the PI-PLC inhibitor U73122 (1 and 3 M). It was found that U73122 but not the inactive analogue of U73122, U73343 (30 M), or PC-PLC inhibitor D609 (30 M) antagonized the potentiating effect of PGE 2 . Furthermore, U73122, U73343, and D609 had no effect on the basal level of Fn expression (Fig. 4). The quantitative data are shown in Fig. 4, lower panels. Because PGE 2 -induced Fn expression was inhibited by U73122, the involvement of the PI-PLC pathway, which increases diacylglycerol levels leading to the activation of PKC, was indicated. The PKC inhibitor GF109203X was thus used to exam- ine whether PKC is involved in the action of PGE 2 . Pretreatment with GF109203X (1-10 M) concentration-dependently inhibited the enhancement effect of PGE 2 (Fig. 5A). PKC isozymes, including ␣, ␤, ⑀, and ␦, have been identified in osteoblasts (27). To examine which PKC isoforms are involved in the potentiation of Fn fibrillogenesis by PGE 2 , isoform-specific AS-ODN was used (23). It was demonstrated that treatment with AS-ODN of the PKC isoform ␣ but not ␤, ⑀, and ␦ antagonized the potentiating action of PGE 2 using ELISA analysis (Fig. 5B). We also directly measured the PKC␣ translocation in response to PGE 2 . Incubation of osteoblasts with PGE 2 (3 M) for 10 or 15 min increased membrane translocation of PKC␣. Pretreatment of osteoblasts for 30 min with SC19220 (10 M) or U73122 (3 M) markedly attenuated the PGE 2induced PKC␣ translocation (Fig. 5C). We then investigated the role of Src in mediating PGE 2 -induced Fn expression using the specific Src inhibitor PP2. As shown in Fig. 6A, PGE 2induced Fn expression was markedly attenuated by pretreatment of cells for 30 min with PP2 (1-10 M) in a concentrationdependent manner. To confirm directly the crucial role of Src in Fn expression, we measured the level of Src phosphorylation in response to PGE 2 . As shown in Fig. 6B, treatment of osteoblasts with PGE 2 (3 M) for 15 min increased c-Src activity, as assessed by immunoblotting samples for phosphotyrosine immunoprecipitated from lysates using c-Src (Fig. 6B). To determine the relationship among the EP 1 receptor, PLC, PKC, and Src in the PGE 2 -mediated signaling pathway, we found that pretreatment of cells for 30 min with SC19220 (10 M), U73122 (3 M), GF109203X (10 M), and PP2 (10 M) markedly inhibited the PGE 2 -induced c-Src activity (Fig. 6B). ELISA measurements also showed that pretreatment of osteoblasts with the U73122 (3 M), GF109203X (10 M), and PP2 (10 M) but not U73343 (30 M) or D609 (30 M) antagonized the Fn upregulation effect of PGE 2 (Fig. 6C). Based on these results, it appears that PGE 2 acts through EP 1 receptor, PLC, PKC, and c-Src-dependent signaling pathway to enhance Fn fibrillogenesis in osteoblasts.
Effect of PGE 2 on the Distribution of Integrin-The assembly of extracellular Fn matrix underneath the cells may be related to integrins (9). Integrins are a family of heterodimeric transmembrane receptors containing ␣ and ␤ subunits. The different combination of ␣ and ␤ chains forms different receptors for various kinds of ECM molecules. ␣5␤1 integrin is a specific receptor for Fn. Flow cytometry was used to investigate the effect of PGE 2 on the cell surface expression of integrins. As shown in Fig. 7A, incubation with PGE 2 (3 M) for 24 h significantly enhanced the fluorescence intensity of ␣5 and ␤1 integrins. The increase of cell surface expression of integrins by PGE 2 was antagonized by SC19220 (10 M), U73122 (3 M), GF109203X (10 M), and PP2 (10 M). We thus examined the effect of PGE 2 on the mRNA levels of ␣5 and ␤1 integrins. Cells treated with PGE 2 (3 M) for 6 h increased the mRNA expression of ␣5 and ␤1 integrins, which was antagonized by pretreatment of EP 1 AS-ODN but not by AS-ODN of EP 2 -EP 4 (Fig. 7C). The increase of mRNA expression of integrins by PGE 2 was also antagonized by SC19220 (10 M), U73122 (3 M), GF109203X (10 M), and PP2 (10 M) (Fig. 7D).
Increase of Fn Promoter Activity by PGE 2 -To study further the involvement of the EP 1 receptor, PI-PLC, PKC, and c-Srcdependent pathway in the action of PGE 2 -induced Fn expression, transient transfection was performed using the rat Fn promoter-luciferase constructs, pGL2F1900-Luc, which contain the rat FN gene between positions Ϫ1908 and ϩ136 fused to the luciferase reporter gene. Osteoblasts incubated with PGE 2 (3 M) led to a 3.8-fold increase in Fn promoter activity. The increase of Fn activity by PGE 2 was antagonized by SC19220 (10 M), U73122 (3 M), GF109203X (10 M), and PP2 (10 M) (Fig. 8A). In cotransfection experiments, the increase of Fn promoter activity by PGE 2 was inhibited by EP 1 AS-ODN, but not by AS-ODN of EP 2 -EP 4 (Fig. 8B). Increase of Fn promoter activity by PGE 2 was also inhibited by the dominant negative mutants of PKC␣ and c-Src (Fig. 8C). Taken together, these data suggest that the activation of EP 1 /PI-PLC/PKC␣/c-Src pathway is required for the increase of Fn by PGE 2 in rat osteoblasts.

PGE 2 Enhanced Fn Formation and Bone Volume of Tibia in
Young Rat-Trabecular bone is composed of a lattice or network of branching bone spicules. The spaces between the bone spicules contain bone marrow. PGE 2 and 17-phenyl trinor PGE 2 (30 M, 10 l, once per day) were locally administered into tibia for 7 consecutive days via the implantation of a needle cannula (22-gauge) in young rats weighing 73-88 g, and the rats were sacrificed later on day 14. The vehicle was injected into the contralateral side for comparison. Compared with the vehicle-injected side (Fig. 9A, arrow, shows the hole of the injection site), PGE 2 and 17-phenyl trinor PGE 2 significantly increased the bone volume of the secondary spongiosa (Fig. 9A). Trabecular bone in the secondary spongiosa increased by 91.3 and 81.7% after local administration of PGE 2 and 17-phenyl trinor PGE 2 . The immunohistochemistry also showed that Fn and ␣5␤1 integrin predominantly localized around the trabecular bone. Long term administration of PGE 2 and 17-phenyl trinor PGE 2 increased the staining of Fn, ␣5␤1 integrin, and type I collagen (Fig. 9, B-D). In addition, BMD and BMC increased after application of PGE 2 and 17-phenyl trinor PGE 2 (Table I). DISCUSSION PGEs are among the most potent regulators of bone cell function (28). It is generally accepted that prostaglandins are mediators in bone metabolism. Among the various prostaglandins, PGE 2 is the most important in bone formation and resorption. In a recent study, Mo et al. (29) demonstrated that PGE 2 treatment increases trabecular bone mass in rats. Extensive studies have demonstrated that PGE 2 has both anabolic and catabolic effects on osteoblasts. The results from this study provide evidence that PGE 2 also regulates Fn fibrillogenesis in cultured rat osteoblasts. In the present study, immunocytochemistry, ELISA, and Western blotting analysis were used to investigate the effect of PGE 2 on Fn assembly. The Fn network is an important factor for the differentiation, expression of physiological function, and survival of osteoblasts (30).
Here we further identify Fn as a target protein for the PGE 2 signaling pathway that regulates cell survival and differentiation. We also show that potentiation of Fn fibrillogenesis by PGE 2 requires an activation of the EP 1 receptor, PI-PLC, PKC␣, and c-Src signaling pathway. PGE 2 stimulated Fn fibrillogenesis in a concentration-dependent manner as detected by immunocytochemistry and ELISA. Furthermore, PGE 2 increased the protein levels of Fn as demonstrated by Western blotting analysis. PGEs, acting through different cell surface receptors on osteoblastic cells, stimulate bone remodeling by promoting both anabolic and catabolic responses, the relative responses being dependent on the target cell population and the concentration of PGE 2 . However, we demonstrate that the EP 1 but not other EP receptors was required for PGE 2 -induced Fn formation. Treatment with butaprost (EP 2 agonist), sulprostone (EP 3 agonist), and 11deoxy-PGE 1 (EP 2 /EP 4 selective agonist) failed to up-regulate Fn expression (Fig. 3A). Furthermore, we could not inhibit PGE 2 -induced Fn up-regulation by EP 2 , EP 3 , and EP 4 receptorspecific antisense oligonucleotides (Fig. 2B). It has been reported that sulprostone also acts on the rat EP 1 receptor (26).
Here we found that sulprostone did not increase Fn expression unless at a high concentration of 20 M. Pretreatment of osteoblasts with EP 1 AS-ODN but not EP 3 AS-ODN antagonized the increase of Fn by 20 M sulprostone. These results indicate that sulprostone also activates the EP 1 receptor at higher concentrations in osteoblasts, which is consistent with the result of vascular endothelial growth factor-C expression in lung cells (31). EP 1 receptor antagonist significantly suppressed PGE 2induced Fn formation, suggesting that EP 1 receptor-dependent pathway is involved in Fn up-regulation by PGE 2 . EP 1 receptor is coupled to Ca 2ϩ mobilization (15), and the intracellular free calcium chelator (BAPTA-AM) antagonized the up-regulation of Fn by PGE 2 . In addition, PGE 2 and 17-phenyl trinor PGE 2 also increase fluorescence intensity of fluo-3. The increase of [Ca 2ϩ ] i may be attributable to the activation of PGE 2 through the EP 1 receptor.
Several isoforms of PKC exist in primary cultured osteoblasts, including ␣, ␤, ⑀, and ␦ (27). Treatment with antisense oligonucleotides directed against the PKC␣ isoform but not PKC␤, -⑀, and -␦ antagonized the potentiating action of PGE 2 in Fn expression, indicating that the ␣ isozyme is much more important to mediate the action of PGE 2 in osteoblasts. We demonstrated that the PKC inhibitors GF109203X antagonized the PGE 2 -mediated potentiation of Fn expression in a dose-dependent manner, suggesting that PKC activation is an obligatory event in PGE 2 -induced Fn expression in these cells. This was further confirmed by the result that the dominant negative mutant of PKC␣ inhibited the enhancement of Fn promoter activity by PGE 2 . PKC is activated by the physiological activator, diacylglycerol, which can be generated either directly, by the action of PLC, or indirectly, by a pathway involving the production of phosphatidic acid by PLD, followed by a dephosphorylation reaction catalyzed by phosphatidate phosphohydrolase. The PLC involved in the production of diacylglycerol is PI-PLC or PC-PLC (32,33). The PI-PLC inhibitor U73122 inhibited PGE 2 -induced Fn expression, whereas the PC-PLC inhibitor D609 and the inactive U73122 analogue U73343 did not affect the action of PGE 2 .
The cytoplasmic protein-tyrosine kinase c-Src was found to be activated by PGE 2 in osteoblastic cells (34). These effects were inhibited by GF109203X, indicating the involvement of PKC-dependent c-Src activation in PGE 2 -mediated Fn induction. In addition to gene expression, a similar signal pathway has also been reported in the development of ischemic preconditioning in the conscious rabbit, which involved PKC⑀-dependent Src and Lck activation (35), in the G protein-coupled receptors regulating N-methyl-D-aspartic acid receptor in CA1 pyramidal neurons, which involved PKC-dependent c-Src activation (36), and in the cellular response to oxidative stress, which involved PKC␦-dependent c-Abl activation (37). Taken together, our results provided evidence that PGE 2 up-regulates Fn in rat osteoblasts via the EP 1 /PI-PLC/PKC␣/c-Src signaling pathway.
Direct osteoblast interactions with the extracellular matrix are mediated by a selective group of integrin receptors includ- FIG. 9. PGE 2 increased bone volume and immunostaining of Fn, ␣5␤1 integrin, and type I collagen in tibia metaphysis of rats. PGE 2 or 17-phenyl trinor PGE 2 (30 M, 10 l, once/day) was locally administered into the tibia through the needle cannula (arrow) in the proximal tibia for 1 week. Vehicle was injected into the contralateral side for comparison. Rats were sacrificed, and the tibiae were used for the analysis of bone volume 7 days after the last injection. Compared with vehicle-treated side, chronic treatment with PGE 2 or 17-phenyl trinor PGE 2 markedly increased bone volume (A). Immunostaining showed that Fn predominantly localized around the trabecular bone (arrowhead) and PGE 2 or 17-phenyl trinor PGE 2 increased the staining of Fn (B), ␣5␤1 integrin (C), and type I collagen (D). Bars ϭ 0.5 mm (A) and 100 m (B-D).

TABLE I
Effect of PGE 2 and 17-phenyl trinor PGE 2 on the bone mineral density, bone mineral content and bone volume in tibia PGE 2 and 17-phenyl trinor PGE 2 (30 M, 10 l, once/day) were locally administered into the tibia by a needle cannula in the proximal tibia for 1 week. Vehicle was injected into the contralateral side for comparison. Rats were sacrificed, and the tibiae were used for analysis 7 days after the last injection. The abbreviation used is as follows: BV/TV, bone volume/tissue volume. ing ␣5␤1, ␣3␤1, ␣v␤3, and ␣4␤1 (38,39). ␣5␤1 integrin, a specific Fn receptor, mediates critical interactions between osteoblasts and Fn required for both bone morphogenesis and osteoblast differentiation (19). Interfering with interactions between Fn and integrin Fn receptors in immature fetal rat calvarial osteoblasts suppressed formation of mineralized nodules in vitro and delayed expression of tissue-specific genes, including osteocalcin (19). The finding that enhancement of surface expression of ␣5 and ␤1 integrins by PGE 2 correlated the increase of Fn assembly by PGE 2 . Increase of the surface expression of ␣5 and ␤1 integrin by PGE 2 was also antagonized by SC19220, U73122, GF109203X, and PP2, suggesting that the regulation of ␣5 and ␤1 integrin expression is parallel to the increase of Fn assembly. PGEs are considered important local factors that modulate bone metabolism through their effects on osteoblastic cells and osteoclasts (12). The skeleton is an important target tissue for PGE 2 , which is involved in bone development, growth, remodeling, and repair (40). By using local injection of PGE 2 and 17-phenyl trinor PGE 2 into the tibia for 7 consecutive days, we demonstrate that local administration of PGE 2 and 17-phenyl trinor PGE 2 increased the bone volume and immunostaining of Fn, ␣5␤1 integrin, as well as type I collagen in young rats. The present results suggest that PGE 2 plays an important role in the developing bone as well. The increase of bone formation may also be partially mediated by the increase of proliferation and survival of osteoblasts, because PGE 2 also increased the differentiation marker of bone sialoprotein (41). Local injection of PGE 2 and 17-phenyl trinor PGE 2 also increased BMD and BMC in young rats, indicating that PGE 2 plays an important role in the regulation of bone formation via the EP 1 receptor. We injected high concentrations of drugs in small volumes in the in vivo studies. Therefore, the action of the EP 1 agonist on the other EP receptors cannot be excluded.
In conclusion, the signaling pathway involved in PGE 2 -induced Fn expression in rat osteoblasts has been explored. PGE 2 increases ␣5 and ␤1 integrins and Fn expression by binding to the EP 1 receptor and activation of phospholipase C, PKC␣, and c-Src. Local administration of PGE 2 and EP 1 agonist increases Fn and promotes bone formation in rat.